Fixation time

Ghazani AA, Arneson NC, Warren K, et al. Limited tissue fixation times and whole genomic amplification do not impact array CGH profiles. J. Clin. Pathol. 2006 59 311-315. 

There are a number of different cell fixation and process methods used in laboratories worldwide. Fixation time in tissue will reflect a commonly accepted fixation time such as that seen in pre-analytical guidelines published in the package inserts for commercially available kits. Regarding cell lines points to note are how soon are the cells fixed after harvesting, are the cells fixed in suspension, or when are they in a suspension matrix such as agarose. 

Finally temperature and tissue type do have an effect on the penetration and fixation. This, combined with a failure to appreciate protein cross-linking time or actual fixation time required for formaldehyde, is probably the main reason for intra- and interlaboratory variation. 

Shorter fixation times produce substantially greater variability across specimens in the same laboratory and even more so across laboratories. At the... 

Standardization of IHC/ICC has been a critical issue for more than three decades, especially with the advances in targeted therapy such as the development of trastuzumab (Herceptin) for advanced breast cancer.51 Nevertheless, standardization is a difficult issue because numerous factors may influence the consistency and reliability of immunostaining results, including fixatives, fixation time, AR, antibody clones, detection system, and interpretation (see Part II). In cytopathology, the situation is even worse due to its variable cell sample preparation techniques. Cytopreparation is. .. the foundation of cytomorphology. 52 We believe it is also the foundation of ICC. Therefore, standardization of ICC needs to start with uniform and reliable cytopreparation. 

As part of this work, we evaluated the effect of fixation times, from Oh to 14 days, on shotgun proteomic analyses and found no significant differences (Fig. 20.8). 

Figure 20.8 Identifications of spectral counts, peptide sequences, and proteins in archival FFPE liver tissue across a time course of increasing fixation time. Reproduced with permission from Reference 20.

The range of doses used at the repeat fixation time can be those which induce a suitable degree of mitotic inhibition at the earlier fixation time, but if the highest dose reduces the MI to an unacceptably low level at the second sampling time, the next highest dose should be chosen for screening. 

A complete assay requires the test material to be investigated at a minimum of three doses together with a positive (untreated) and solvent-only control can be omitted if tissue culture medium is used as a solvent. When two fixation times are used in repeat tests, the positive control is necessary at only one time but the negative or solvent control is necessary at both times. 

Storing and titrating antibodies correctly are often easy solutions when no staining is observed. Some antibodies are extremely sensitive to repeat freeze-thaw cycles and others have a limited shelf life, as evidenced by a short expiry dates, ft is important to check the compatibility of the primary and secondary antibodies before starting IHC, as using the wrong secondary IgG is a common but easily corrected problem. As mentioned, antigen retrieval can be a problem when tissue has been overfixed, so special attention should be paid to fixation time and, once optimized, should be held constant for all subsequent runs. 

Formaldehyde works well as a primary fixative in this setting because it preserves cell morphology well and the time of exposnre to the fixative is short. Formaldehyde fixation at room temperatnre is very effective, bnt at 4°C, it is a very poor fixative. Longer fixation times (30-60 min) may be helpful for some antigens that are difficult to preserve. 

Longer fixation times may be necessary to fix the suspension grown cells adequately to the plate. 

Reduce overestimation and fixation time on a perceptual task. 

Battifora, H. 1991. Effect of fixatives and fixation times on tissues. Am. J. Clin. Pathol. 96 144. 

Daidone, M. G., Benini, E., Rao, S., Pilloti, S., and Silvestrini, R. 1998. Fixation time and microwave oven irradiation affect immunocytochemical p53 detection in formalin-fixed paraffin sections. Appl. Immunohistochem. 6 140-144. 

James, J. D., and Hauer-Jensen, M. 1999. Effects of fixative and fixation time for quantitative computerized image analysis of immunohistochemical staining. J. Histotechnol. 22 109-111. 

Mintze, K., Macon, N., Gould, K. E., and Sandusky, G. E., 1995. Optimization of proliferating cell nuclear antigen (PCNA). Immunohistochemical staining A comparison of methods using three commercial antibodies, various fixation times, and antigen retrieval solutions. J. Histotechnol. 78 25-30. 

It is also likely that certain antigens have optimal durations of fixation. It has been shown that for the immunohistological demonstration of estrogen receptor, a minimum fixation time of 6-8 hrs in formalin is required for consistent results (Goldstein et al 2003). This minimum time is particularly critical in small biopsies and needle cores which tend to receive insufficient or weak initial formaldehyde fixation followed by extraction/fixation in ethanols used for dehydration in the tissue processor, resulting in inconsistent demonstration of some tissue antigens. 

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